Abstract

Certain plant viruses encode suppressors of posttranscriptional
gene silencing (PTGS), an adaptive antiviral defense response that
limits virus replication and spread. The tobacco etch potyvirus
protein, helper component-proteinase (HC-Pro), suppresses PTGS of
silenced transgenes. The effect of HC-Pro on different steps of the
silencing pathway was analyzed by using both transient
Agrobacterium tumefaciens-based delivery and transgenic
systems. HC-Pro inactivated PTGS in plants containing a preexisting
silenced β-glucuronidase (GUS) transgene. PTGS in this system was
associated with both small RNA molecules (21–26 nt) corresponding to
the 3′ proximal region of the transcribed GUS sequence and cytosine
methylation of specific sites near the 3′ end of the GUS transgene.
Introduction of HC-Pro into these plants resulted in loss of PTGS, loss
of small RNAs, and partial loss of methylation. These results suggest
that HC-Pro targets a PTGS maintenance (as opposed to an initiation or
signaling) component at a point that affects accumulation of small RNAs
and methylation of genomic DNA.

Posttranscriptional gene
silencing (PTGS) or RNA interference occurs in a wide variety of
organisms, including plants, animals, and fungi (1, 2). The PTGS
process involves recognition of a target RNA and initiation of a
sequence-specific RNA degradation pathway in the cytoplasm. Targets for
PTGS may be recognized because of the presence of extensive
double-stranded RNA (dsRNA) structure or because of an aberrant feature
of the RNA (1). Small RNAs of 21–23 nt, corresponding to both sense
and antisense strands of the target, are consistently associated with
PTGS (2–6). It was proposed that these short RNAs provide specificity
for target RNA degradation through association with an RNaseIII-like
enzyme (2, 6). In plants, PTGS of transgenes is typically associated
with methylation of nuclear DNA corresponding to the transcribed region
of the target RNA, although transcription levels of the transgene are
generally unaffected (1). In addition, systemic signaling to trigger
PTGS at a distance can occur in plants, presumably through transport of
a nucleic acid signal (7–9). Several genes encoding factors required
for PTGS or RNA interference have been isolated, and these include
proteins with similarities to an RNA-dependent RNA polymerase (5, 10,
11), a RecQ-like DNA helicase (10), an RNaseD-like protein (12), and a
protein encoded by the
piwi/sting/argonaute/zwille/eIF2C
gene family (13).

Although PTGS in plants has been studied most extensively by using
transgenes, viruses are both initiators and targets of PTGS. Infection
by a range of viruses results in PTGS-like responses, even in the
absence of homologous nuclear sequences (14–17). Virus-induced PTGS
presumably reflects an adaptive defense mechanism whereby a
sequence-specific response limits the extent of virus infection. Among
the early indications that the PTGS response could have antiviral
effects was the discovery that several plant viruses encode a PTGS
suppressor (18–21). The potyviruses encode helper component-proteinase
(HC-Pro), a multifunctional protein required for maintenance of genome
replication, long-distance movement through plants, and polyprotein
processing (22–24). Expression of HC-Pro in plants by either a
transgene or a virus vector is sufficient to inhibit PTGS of a
transgene (19–21). It was proposed that the PTGS-suppressing activity
of HC-Pro accounts for the requirement of HC-Pro in replication and
long-distance movement (19).

In this article, the effects of HC-Pro on specific steps in the PTGS
process were determined. The results show that HC-Pro partially
reverses silencing in cells with a preexisting silenced transgene,
suggesting that HC-Pro inhibits a function required for maintenance of
the silenced state. Data also reveal that HC-Pro acts to inhibit a step
upstream from production of short RNAs in the silencing pathway and to
reduce silencing-associated transgene methylation.

Materials and Methods

Transgenic Plants and Plasmids.

Nicotiana tabacum plants expressing a nontranslatable
β-glucuronidase (GUS) gene under the control of the 35S promoter and
terminator have been characterized previously (Fig.
1A; ref. 19). The homozygous
transgenic lines 407 and 422 are posttranscriptionally GUS-silenced,
whereas the 446 line contains the same transgene but is nonsilenced.
The transgenic line U-6B contains a polyprotein including the P1
proteinase, HC-Pro, and the N terminus of the P3 protein and has been
described (25). Several lines were derived from crosses between the 407
GUS-silenced line and the U-6B P1/HC-Pro-expressing line (19). A
brief description of the plants used in this study is provided in Table
1.

The plasmids pRTL2-GUS and pRTL2–0027 expressing the GUS and tobacco
etch virus P1/HC-Pro coding sequences, respectively, have been
described (25, 26). These plasmids contain the 35S promoter and
terminator sequences. The plasmid pSLJ755I5-GUS was constructed by
inserting the expression cassette from PstI-digested
pRTL2-GUS into the binary plasmid pSLJ755I5 (27). The dual expression
plasmid, pSLJ755I5-GUS + HC-Pro, was constructed by insertion of the
HindIII-digested expression cassette from pRTL2–0027 into
pSLJ755I5-GUS. The resulting plasmid, pSLJ755I5-GUS + HC-Pro, contained
the GUS and tobacco etch virus P1/HC-Pro coding sequences under
the control of independent 35S promoters (Fig. 1B). The
three binary plasmids, pSLJ755I5, pSLJ755I5-GUS, and pSLJ755I5-GUS +
HC-Pro, were referred to as vector, GUS, and GUS + HC-Pro plasmids,
respectively. Each of these plasmids was introduced by triparental
mating into the virulent Agrobacterium strain GV2260 and the
avirulent strain C58C1D.

Agrobacterium Injection.

Individual Agrobacterium colonies were grown for 20 h
in 5-ml cultures (Luria broth, 100 μg/ml rifampicin, 12.5
μg/ml tetracycline) at 30°C. This was used to inoculate a
50-ml culture (Luria broth, 20 μM acetosyringone/10 mM Mes, pH
5.7/12.5 μg/ml tetracycline), which was grown for 16–20 h at
30°C. The bacteria were pelleted by centrifugation, resuspended in
infiltration medium (10 mM MgCl2/10 mM
Mes, pH 5.7/150 μM acetosyringone) to 0.5 OD at 600 nm, and
incubated at room temperature for a minimum of 3 h (28). By using
a 3-ml syringe, the Agrobacterium solution was injected into
leaves through an incision.

For silencing-release assays, two zones on apposing half-leaves of
either nontransgenic (line 13) or GUS-silenced (line 7) tobacco plants
were injected with combinations of Agrobacterium containing
empty vector, GUS, or GUS + HC-Pro plasmids. Leaves were harvested at 4
days after injection and GUS activity was visualized by infiltration
with the colorimetric substrate, 5-bromo-4-chloro-3-indolyl
β-D-glucuronide. After overnight incubation at
room temperature, the leaves were cleared in 75%
(vol/vol) ethanol at 70°C. Leaves were photographed, and
images were processed electronically by using Adobe
photoshop.

Nucleic Acid Isolation.

Total RNA was extracted from mature tobacco leaves (2.0 g) as described
(19). High-molecular weight RNA was precipitated in the presence of 2 M
lithium chloride. Low-molecular weight RNA was precipitated from the 2
M lithium chloride supernatant in the presence of 75% (vol/vol)
ethanol, resuspended in sterile water, and reprecipitated in the
presence of 70% (vol/vol) ethanol and 0.1 M sodium acetate. The
precipitate was washed with 70% (vol/vol) ethanol and
resuspended in 40 μl of diethyl pyrocarbonate-treated deionized
water. Alternatively, small RNAs were isolated by anion exchange
chromatography (RNA/DNA Midi Kit; Qiagen, Chatsworth, CA) after
removal of large RNA by precipitation with 2 M lithium chloride and DNA
by precipitation in 6% (vol/vol) polyethylene glycol 8000 and
0.8 M sodium chloride.

Genomic DNA was isolated as described (19) and digested with
restriction endonucleases in 16-h reactions. The DNA was extracted from
the reaction mixture by using phenol/chloroform/isoamyl
alcohol, precipitated in the presence of 70% (vol/vol) ethanol
and resuspended in 20 μl of sterile deionized water.

Gel Blot Analysis.

High molecular weight RNA (10 μg) and genomic DNA (20 μg) samples
were subjected to blot hybridization analysis, as described (19).
Radiolabeled probes for specific GUS sequences were made by random
priming reactions in the presence of [32P]dATP
(29), and radioincorporation was measured. RNA blots were stripped and
reprobed by using a tobacco ribosomal RNA probe labeled with
[32P]dATP, and DNA blots were reprobed by using
a tobacco eIF4E probe labeled with [32P]dATP
(30).

Results

HC-Pro Suppresses the Maintenance Phase of PTGS.

Previous analyses of silencing suppression by HC-Pro depended on
delivery of HC-Pro into plants through either a stable transgene or an
RNA virus vector (18–21). However, determination of whether HC-Pro
suppresses an initiation/recognition step, a maintenance step,
or a signaling step in PTGS with either type of delivery system is
complicated by several factors. By using the transgenic system, both
the HC-Pro and silencing loci are present in most or all cells during
development. Suppression of initiation, maintenance, or signaling of
PTGS would result in the same phenotype, namely, lack of silencing in
the mature plant. By using a virus vector, interpretation of
silencing-suppression phenotypes may be influenced or clouded by the
presence of a suppressor encoded by the vector itself.

To enable analysis of PTGS suppression in a tissue-autonomous manner
and in the absence of a virus vector, a transient system was developed
by using Agrobacterium-mediated delivery of HC-Pro and a
silencing reporter into a plant containing a PTGS locus. Transgenic
N. tabacum plants (line 7) containing a
posttranscriptionally silenced, defective GUS gene were produced and
have been characterized (19). The modified 35S-GUS transgene contains
nonsense and frameshift mutations near the 5′ end and does not encode
an active protein (Fig. 1A). Single-gene or
dual-gene expression plasmids encoding GUS alone (GUS) or GUS plus
HC-Pro (GUS + HC-Pro), respectively, were produced and introduced into
Agrobacterium. The HC-Pro construct actually encoded a
larger region of the tobacco etch virus polyprotein, including the P1
proteinase adjacent to the N terminus of HC-Pro and part of the P3
protein adjacent to the C terminus (Fig. 1B). The N and C
termini of HC-Pro are normally formed by autoproteolytic cleavage of
the viral polyprotein by P1 proteinase and HC-Pro, respectively (31,
32). Therefore, the cassette was predicted to yield mature, accurately
processed HC-Pro after expression in plant cells (see below).

To confirm that the 35S-GUS expression cassettes in the single and dual
vectors were functional, cultures of Agrobacterium
containing the expression plasmids or empty vector were injected into
leaves of nontransgenic N. tabacum plants (line 13). In all
experiments, leaves were injected with combinations of experimental and
control cultures on apposing half-leaves (Fig. 1C) and
infiltrated with a GUS colorimetric substrate at 4 days after
injection. Tissues receiving each plasmid with a 35S-GUS expression
cassette, but not empty vector, generated GUS activity (Fig.
2A). To rule out that the GUS
activity was caused by expression in bacteria, the experiment was done
with both T-DNA transfer-competent (Vir+) and
T-DNA transfer-defective (Vir−) strains of
Agrobacterium. GUS activity was detected only in tissues
injected with Vir+ bacteria containing 35S-GUS
cassettes (Fig. 2B). Based on microscopic
examination, virtually all cells within the infiltration zone contained
GUS activity, suggesting that the Agrobacterium-mediated
delivery of T-DNA in this system was extremely efficient.

Suppression of PTGS by transient Agrobacterium-mediated
delivery of HC-Pro. GUS encoded by single-GUS or dual-GUS + HC-Pro
cassettes was detected by histochemical assay in leaf tissue at 4 days
after infiltration. (A) Control series of
Agrobacterium injection assays with nontransgenic plants
(line 13). (B) Dependence of GUS activity on delivery of
T-DNA by Agrobacterium. Plasmids were introduced into
Vir+ and Vir− strains of
Agrobacterium, followed by injection into nontransgenic
plants. (C) Agrobacterium injection
assays with the same series shown in A, but with
GUS-silenced plants (line 7). Note that GUS activity occurs only in
leaves infiltrated with Agrobacterium containing the
dual GUS + HC-Pro plasmid.

The GUS and GUS + HC-Pro expression cassettes were then introduced into
leaves of silencing line 7. No GUS activity was detected in half-leaves
receiving the vector alone or the single-gene 35S-GUS cassette (Fig.
2C), indicating that the GUS-silencing phenotype of line 7
was maintained after introduction of a functional GUS gene. However,
GUS activity was detected in half-leaves injected with
Agrobacterium containing the dual GUS + HC-Pro plasmid (Fig.
2C), although the intensity of the histochemical signal
generally was less than in leaves of nontransgenic line 13 plants.

To analyze further GUS and HC-Pro in the silencing and nonsilencing
lines after Agrobacterium-mediated gene transfer, tissue
from the injection zone was excised and subjected to immunoblot
analysis by using anti-HC-Pro and anti-GUS sera. In nontransgenic
tissue, GUS accumulated after injection of Agrobacterium
containing either the single or dual expression cassette (Fig.
3, lanes 3–6), whereas HC-Pro
accumulated only in tissue receiving the dual cassette (Fig. 3, lanes 5
and 6). In tissue from silencing line 7, no GUS protein was detected
after introduction of the single 35S-GUS cassette (Fig. 3, lanes 9 and
10). In contrast, GUS protein was detected in tissue receiving the GUS
+ HC-Pro construct, although, like the histochemical assay, the level
of accumulation of GUS in tissue from line 7 was significantly lower
than in tissue from the nonsilencing line 13 (Fig. 3, lanes 11 and 12).
These data indicate that PTGS can be inactivated by HC-Pro, at least
partially, at a postrecognition/postinitiation step in a cell-
or tissue-autonomous fashion that does not require systemic signaling.
These results suggest that HC-Pro suppresses the maintenance phase in
the PTGS process.

Immunoblot analysis of GUS and HC-Pro after
Agrobacterium-mediated delivery into GUS-silenced and
nontransgenic plant lines. Normalized, total detergent-soluble protein
extracts were prepared from tissue injected with
Agrobacterium carrying vector alone or plasmids
containing the single-GUS or dual-GUS + HC-Pro expression cassettes.
Lanes 1–6, nontransgenic line 13. Lanes 7–12, GUS-silenced line 7.
Samples consisted of pools of tissue from four injection zones. Two
samples are shown for each treatment. Immunoblot results with
anti-HC-Pro (Upper) and anti-GUS sera
(Lower) are shown.

HC-Pro Suppresses a Step Before Accumulation of Short RNAs.

To determine whether PTGS of the nontranslatable GUS gene in transgenic
lines 422 and 407 [the parent from which the GUS-silencing locus was
derived in line 7 (Figs. 2 and 3)] was associated with small RNAs,
low-molecular weight RNA was extracted and analyzed by blot
hybridization with a radiolabeled GUS probe. A discrete band of
material that migrated between the 21- and 26-nt single-stranded DNA
standards was detected in GUS PTGS lines 422 and 407 in independent
experiments (Fig. 4A, lanes 2
and 6). No such species was detected in nontransgenic plants (Fig.
4A, lanes 1 and 5) or in plants from line 446 (Fig.
4A, lanes 3 and 7), which contains the same GUS
transgene as in lines 407 and 422 but does not display PTGS (19). The
GUS-related nucleic acid species was sensitive to RNaseA but
insensitive to DNaseI (data not shown), indicating that the material
was RNA. By using probes normalized for total radioactivity and RNA
extracts from line 407, the small RNAs hybridized preferentially to
sequences corresponding to the 3′ proximal region of the GUS coding
sequence, with the most intense hybridization occurring with a probe
for the 3′ terminal 165 nucleotides (Fig. 4B). Little or no
hybridization with small RNAs from line 407 was detected by using
probes representing the 5′ proximal 787 nucleotides of the GUS
sequence. The GUS sequence-related RNAs likely correspond to the
PTGS-specific small RNAs identified by Hamilton and Baulcombe (3).

Detection of short RNAs in GUS-silenced transgenic plants. Low
molecular weight RNA was extracted from leaves of either nontransgenic
(NT), GUS-silenced (422 and 407), or GUS-nonsilenced (446) plants.
Equal amounts of each RNA sample were subjected to electrophoresis in
denaturing 15% polyacrylamide gels, stained with ethidium bromide,
blotted to a nylon membrane, and hybridized using various
32P-labeled GUS DNA fragments as probes. The PTGS status of
each plant is indicated above the lanes. (A) Two
experiments analyzing small RNAs with a full-length,
32P-labeled GUS probe. In vitro transcribed
GUS RNA was hydrolyzed (OH−) and used as a hybridization
control. The arrow indicates the position of short RNA.
(Right) Ethidium bromide staining of the gel used in
experiment 2. DNA oligonucleotides (21, 26, and 32 nt) were used as
standards (STD). (B) Analysis of small RNAs with
normalized (2 × 106 cpm) 32P-labeled
probes corresponding to different regions of the GUS coding sequence.
The positions of the 21 and 26 nt DNA standards are shown at the
right.

To test the effect of HC-Pro on accumulation of short RNAs associated
with PTGS of the GUS sequence, F3 progeny plants
from a cross between the 407 line and a transgenic plant expressing the
P1/HC-Pro region of the tobacco etch virus genome were analyzed.
Plants from the F2 and F3
generations were characterized with respect to transgene configuration
and GUS-silencing phenotype (Table 1). Progeny plants containing either
no transgenes [referred to as nontransgenic (NT) or line 13] or a
homozygous GUS-, HC-Pro-null transgene configuration (line 7) were
nonsilenced or GUS-silenced, respectively (Fig. 2). In addition, an
F2 plant that was homozygous at the GUS transgene
locus and hemizygous at the HC-Pro transgene locus (line 17a) was
identified. The F3 progeny from line 17a were all
homozygous at the GUS-silencing locus but either lacked the HC-Pro
transgene (line 17, silenced) or were homozygous/hemizygous at
the HC-Pro locus (line 17HC, silencing suppressed). Both high and low
molecular weight RNAs were isolated from leaf tissue of NT, 407
parental, line 17, and line 17HC plants and subjected to blot
hybridization by using a GUS-sequence probe. As shown previously (19),
the level of GUS mRNA in silencing-suppressed line 17HC plants was
considerably higher than the levels in GUS-silenced 407 and line 17
plants (Fig. 5A). In contrast,
the level of small RNAs in line 17HC plants was considerably lower than
the levels in 407 or line 17 plants. This result was observed
consistently in multiple experiments, two of which are shown in Fig.
5B. These data indicate that silencing suppression by HC-Pro
blocks accumulation of silencing-specific small RNAs.

HC-Pro suppresses accumulation of short RNAs. (A) Blot
analysis of GUS mRNA in either nontransgenic (NT), GUS-silenced (407
and 17), or silencing-suppressed (17HC) plants. High-molecular weight
RNA was isolated, normalized (10 μg/lane), subjected to
electrophoresis, blotted to a nylon membrane, and hybridized by using
32P-labeled full-length GUS DNA as a probe. The blot was
stripped and reprobed with a 32P-labeled DNA probe specific
for rRNA. The positions of both GUS and rRNA mRNAs are indicated.
(B) Blot analysis of short RNA. Low-molecular weight RNA
was isolated and analyzed as described in the legend for Fig. 4. The
results from two independent experiments (Expt.) are shown. The arrow
indicates the position of silencing-specific short RNA. For
presentation purposes, the data shown in A and
B are composite images from noncontiguous lanes from a
single blot.

Previous experiments revealed that delivery of HC-Pro into plants
containing a posttranscriptionally silenced reporter gene does not
reverse cytosine methylation associated with the transcribed sequence
of the transgene (33). These observations were based on introduction of
HC-Pro with an RNA virus vector, when PTGS of the transgene was already
operative, and cells at the time of inoculation contained previously
methylated DNA. To determine whether the presence of HC-Pro affects
methylation of a GUS-silencing locus during development and through
multiple generations, the cytosine-methylation status of the GUS
transgene in 446, line 7, line 17, and line 17HC plants was analyzed by
using methylation-sensitive restriction enzymes followed by DNA blot
hybridization. Most methylation-sensitive restriction enzymes tested,
however, did not reveal consistent methylation patterns in the
GUS-silenced line 7 and line 17 plants (data not shown). Only
HaeIII revealed consistent cytosine methylation of the GUS
sequence in line 7 and line 17 plants, as revealed by the appearance of
partial-digestion products (Fig.
6B, lanes 3 and 4). Three DNA
fragments (2,166, 1,821, and 849 nt) were identified as partial
digestion products containing 3′ proximal-GUS coding sequences based on
analysis of size and on further hybridization analysis with specific
probes (Fig. 6 and data not shown). These products arose through
inhibition of digestion at HaeIII sites H4 and/or H5
(Fig. 6A). No partial digestion products
corresponding to those predicted if HaeIII sites H1, H2, or
H3 were methylated were identified. As shown by stripping the blot and
reanalyzing with an eIF4E-specific probe (Fig. 6B, lanes
6–10), all HaeIII sites in a control gene were digested to
completion, indicating that the GUS-transgene results were not simply
caused by insufficient HaeIII reaction conditions. Based on
densitometric scans of blots from three independent experiments and
normalization of results based on the nucleotide length of each
fragment, cytosine residues at approximately 10% and 12% of H4 sites,
and 54% and 40% of H5 sites, were methylated in GUS-silenced lines 7
and 17, respectively.

HC-Pro partially suppresses methylation of target DNA.
(A) Schematic representation of DNA corresponding to the
GUS coding sequence. Positions of HaeIII restriction
sites (H1-H5) and sizes (in nucleotides) of the expected digestion
products are illustrated. Sites marked by an asterisk contain cytosines
in a symmetrical (CpNpG) configuration. Filled circles indicate
HaeIII sites that were cytosine methylated in
GUS-silenced plants. The right (RB) and left (LB) borders of the GUS
transgene are indicated. (B) Blot analysis of genomic
DNA in nontransgenic (NT), GUS-silenced transgenic (7 and 17),
GUS-nonsilenced transgenic (446), and GUS-silencing suppressed (no.
17HC) plants. Blots were hybridized with a 32P-labeled
probe specific for the GUS gene. The blot was stripped and rehybridized
with a 32P-labeled DNA probe specific for the eIF4E coding
sequence.

In contrast to results with GUS-silenced line 7 and line 17 plants,
evidence for methylation was obtained only for site H5 in the GUS
transgene in line 17HC plants (Fig. 6B, lane 5). Results
consistent with cytosine methylation at 20% of H5 sites were obtained
based on averages from three experiments. Similarly, evidence for
cytosine methylation in the nonsilenced 446 GUS transgene was obtained
for a low percentage of H5 sites (8%; Fig. 6B, lane 2).
These results indicate that silencing suppression by HC-Pro results in
a decrease in the extent of silencing-associated methylation of DNA.

Discussion

The point of HC-Pro-mediated suppression in the PTGS pathway was
analyzed by using a series of transient and transgenic assays. These
experiments were done in the absence of a replicating virus vector. The
results, therefore, are free from the potential complications
associated with extraneous suppressors that may be encoded by a virus
vector.

Three conclusions were drawn from these results. First, HC-Pro
suppresses one or more maintenance steps in the PTGS pathway. Transient
delivery of HC-Pro by Agrobacterium injection into tissue of
a plant with a silenced GUS transgene was sufficient to inhibit
silencing in a cell- or tissue-autonomous manner, indicating that
suppression occurs beyond the point of initiation of silencing and
without the need for systemic signaling. In contrast, the cucumber
mosaic virus 2b protein was suggested to suppress a signaling step, but
not a maintenance step, in the silencing pathway (20). The transient
expression data also imply that HC-Pro targets or suppresses a factor
that is required on a continual basis or that is relatively labile.
Silencing suppression by HC-Pro in the transient assay, however, was
not complete. Incomplete silencing suppression could be caused by a
number of factors, including the inability of HC-Pro to suppress
silencing in all cells, a quantitative effect reflecting the activity
of residual silencing factors, or the sampling of tissue at a time
point at which silencing suppression was incomplete.

Second, HC-Pro inhibits a step required for accumulation of small RNAs
in the PTGS pathway. Transgenic plants with a silenced GUS gene
accumulated small RNAs of approximately 21–26 nts in length, which
almost certainly correspond to small RNAs associated with PTGS or RNA
interference identified by others (2–6). By using an in
vitro RNA interference system from Drosophila cells,
Zamore et al. (6) demonstrated that the small RNAs likely
derive from dsRNA inducer molecules rather than from degradation of the
target mRNA. Small RNAs are proposed to be produced by cleavage of a
dsRNA precursor by an RNaseIII-like enzyme, to remain associated with
the nuclease, and to confer sequence specificity to the nuclease (2,
6). Introduction of HC-Pro through a genetic cross substantially
reduced levels of small RNA. The most straightforward interpretation of
this result is that HC-Pro suppresses a step upstream of, or at the
point of, production of small RNAs. Such a step could be recognition of
an inducer dsRNA molecule, modification of an inducer molecule,
interaction of the putative RNaseIII-like nuclease with the dsRNA, or
processing of the dsRNA to form the small RNAs. Alternatively, small
RNA production may depend on a feedback-amplification loop from a point
downstream of initial production of limited quantities of small RNAs.
In this case, HC-Pro could conceivably affect any point in the loop.

Third, HC-Pro reduces the level of cytosine methylation of a transgene
sequence that is a PTGS target. When HC-Pro was expressed from a
transgenic plant with a GUS PTGS locus, the relative level of cytosine
methylation at two HaeIII sites (one symmetrical and one
nonsymmetrical) near the 3′ end of the GUS-transcribed sequence was
lower than in plants containing only the GUS PTGS locus. Methylation at
one site (H5), however, was not eliminated entirely in the presence of
HC-Pro. On the other hand, a low level of methylation also was detected
at the H5 in the GUS transgene locus from plant 446, which did not
display PTGS. The basis for PTGS-associated methylation is not yet
clear, although it likely involves an RNA-mediated feedback mechanism
from the cytoplasm (33). Sites of methylation at the GUS PTGS locus in
line 7 and line 17 plants correlated roughly with the GUS sequence
represented among the small RNAs, because both methylation sites and
small RNAs were associated with 3′ proximal sequences of the transgene
or GUS RNA. One possible interpretation of these results is that
methylation of the PTGS transgene locus is guided by small RNAs that
diffuse from the cytoplasm and interact with chromosomal DNA (1).
Inhibition of small RNA accumulation by HC-Pro, therefore, would lead
to reduced methylation of the transcribed region of the PTGS transgene.

The specific factor or factors in the PTGS pathway that are affected by
HC-Pro remain to be determined. It is possible that HC-Pro interacts
with a PTGS structural or regulatory factor. Structural factors that
are proposed to function upstream of small RNA accumulation in the
pathway include the host RNA-dependent RNA polymerase (5, 34, 35) and a
dsRNA-binding ribonuclease (2, 6). Regulatory factors may influence the
production, activation, or accumulation of functional forms of these
factors. The identities and functions of PTGS pathway components and
the effects of HC-Pro on their activities are key problems to address.

Acknowledgments

We thank Brian Staskawicz and Doug Dahlbeck for supplying
Agrobacterium strains GV2260 and C58C1D and
for helpful comments on setting up the injection system. We thank
Jonathan Jones for providing pSLJ755I5. We are grateful to Julia
Gothard and Sue Vogtman for excellent help in the greenhouses. This
work was supported by United States Department of Agriculture Grant
98-35303-6485 and National Institutes of Health Grants AI 43288 and AI
27832.

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